Eur J Nucl Med Mol Imaging (2009) 36:115–121
DOI 10.1007/s00259-008-0925-8

ORIGINAL ARTICLE

Usefulness of competitive inhibitors of protein binding
for improving the pharmacokinetics of 186Re-MAG3-conjugatedbisphosphonate (186Re-MAG3-HBP), an agent for treatmentof painful bone metastases
Kazuma Ogawa & Takahiro Mukai & Keiichi Kawai &Norito Takamura & Hirofumi Hanaoka &Kazuyuki Hashimoto & Kazuhiro Shiba &Hirofumi Mori & Hideo Saji

Received: 21 December 2007 / Accepted: 28 July 2008 / Published online: 16 August 2008
# Springer-Verlag 2008

Abstract
Purpose We have developed a 186Re-mercaptoacetylglycylglycylglycine complex-conjugated bisphosphonate(186Re-MAG3-HBP) for the treatment of painful bonemetastases. We assumed competitive inhibitors of protein
binding to be useful for procuring a favorable biodistribution of 186Re-MAG3-HBP for the palliation of bone pain
because it has been reported that the concurrent administration of 99mTc-MAG3 and drugs with high affinity forserum protein produced competitive displacement at specific binding sites and enhanced total clearance and tissue
distribution.
Methods The displacement effects of several protein-binding inhibitors on the protein binding of 186Re-MAG3-HBP
were investigated. Biodistribution experiments were perK. Ogawa : T. Mukai : H. Hanaoka : H. Saji (*)
Department of Patho-Functional Bioanalysis,
Graduate School of Pharmaceutical Sciences,
Kyoto University,
Yoshida Shimoadachi-cho, Sakyo-ku,
Kyoto 606-8501, Japane-mail: hsaji@pharm.kyoto-u.ac.jp
K. Ogawa : K. Shiba : H. Mori
Advanced Science Research Center,
Kanazawa University,
13-1 Takara-machi,
Kanazawa 920-8640, JapanT. Mukai
Graduate School of Pharmaceutical Sciences,
Kyushu University,
3-1-1 Maidashi, Higashi-ku,
Fukuoka 812-8582, Japan

formed by intravenously administering 186Re-MAG3-HBP
into rats with ceftriaxone as a competitive protein-binding
inhibitor or saline.
Results The protein binding of 186Re-MAG3-HBP in rat
serum, human serum, and a human serum albumin solution
was significantly decreased by the addition of ceftriaxone,
which has high affinity for binding site I on serum albumin.
In the biodistribution experiments, pretreatment withceftriaxone enhanced the clearance of the radioactivity of186
Re-MAG3-HBP in blood and nontarget tissues but had
no effect on accumulation in bone.
Conclusions The findings suggested that the use of proteinbinding competitive inhibitors would be effective in
improving the pharmacokinetics of radiopharmaceuticals
with high affinity for serum protein.
K. Kawai
Graduate School of Medical Sciences, Kanazawa University,
5-11-80 Kodatsuno,
Kanazawa 920-0942, JapanK. Kawai
Biomedical Imaging Research Center, University of Fukui,23-3 Shimoaizuki, Matsuoka,
Yoshida, Fukui 910-1193, Japan
N. Takamura
School of Pharmaceutical Sciences,
Kyushu University of Health and Welfare,
1714-1 Yoshino,
Nobeoka 882-8508, JapanK. Hashimoto
Japan Atomic Energy Agency,
Tokai-mura, Ibaraki 319-1195, Japan

116

Keywords Protein binding . Absorbed radiation dose .
Palliation . Bisphosphonate . 186Re

Introduction
Bone metastases often present with severe bone pain, which
has a significant impact on patients’ quality of life [1].
Systemic radionuclide therapy using specifically localizedbone-seeking radiopharmaceuticals is preferable because of
the few side effects and long-lasting therapeutic effects
brought about by a single injection [2]. Previously, basedon the concept of bifunctional radiopharmaceuticals, wehave developed 186Re complex-conjugated bisphosphonate
analogs for the palliation of painful bone metastases andhave demonstrated the utility of these compounds [3–7].
Among these compounds, 186Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate,[[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]
carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate] oxorhenium(V) (186Re-MAG3-HBP) showed superior biodistribution characteristics as a therapeutic agent
for the palliation of painful bone metastases [3]. However,
186
Re-MAG3-HBP may have high affinity for serum protein
because 99mTc-MAG3-HBP had a high rate of binding toserum protein, which might be attributed to the high proteinbinding ratio of the 99mTc-MAG3 complex [8–10]. We
synthesized a 99mTc-6-hydrazinopyridine-3-carboxylic
acid (HYNIC)-conjugated bisphosphonate, which had alower protein-binding ratio and improved biodistribution
compared with that of 99mTc-MAG3-HBP [11]. However,since HYNIC cannot form a stable complex with 186Re, inthis study, we tried a different approach to reducing theprotein binding.
The binding of drugs to serum proteins has important
pharmacokinetic consequences because it influences theirdistribution, metabolism, and excretion. Many of the interactions occur in relatively well-defined regions on humanserum albumin (HSA) and on α1-acid glycoprotein (AGP).
Sudlow et al. reported the presence of two primary drugbinding sites on HSA namely, site I (also referred to as thewarfarin-binding site) and site II (the indole-benzodiazepinebinding site) [12, 13]. As a result, coadministration ofdrugs with high protein-binding affinity might produce
competitive displacement at the binding site or might
influence the simultaneous binding of another drug byelectrostatic effects and/or binding-induced conformational changes in the protein, causing higher free concentrations of drugs in plasma than with the administration of
the drugs alone [14–17]. Kawai et al. applied a strategy forthe competitive displacement of protein bindings toradiopharmaceuticals for the first time and reported thattreatment of a drug with high protein binding affinity

Eur J Nucl Med Mol Imaging (2009) 36:115–121

increased the free fraction of 99mTc-MAG3, achieving
rapid accumulation in the kidney and fast clearance fromthe blood [18].
In this study, the displacement effects of several drugs
with high affinity for HSA-binding site I and site II on
186
Re-MAG3-HBP were evaluated in vitro. Biodistribution
experiments of 186Re-MAG3-HBP with or without pretreatment with an inhibitor were performed to investigate
the validity of employing a competitive inhibitor of protein
binding.

Materials and methods
Materials
186

Re was supplied by the Japan Atomic Energy Agency(Tokai-mura, Japan) as 186ReO4− [19]. 186Re-MAG3-HBP
and nonradioactive Re-MAG3 were synthesized as described previously [3]. HSA (essentially fatty acid freealbumin), AGP, ceftriaxone, and cefazolin were purchasedfrom Sigma Chemical (St. Louis, MO, USA). Bucolome (5n-butyl-1-cyclohexyl-2,4,6-trioxoperhydropyrimidine, Grelan Pharmaceutical, Tokyo, Japan) was obtained as a puresubstance from the manufacturer. Ibuprofen was purchasedfrom Nakalai Tesque (Kyoto, Japan). Other reagents wereof reagent grade and used as received.
In vitro protein-binding studies
The protein-binding ratios of 186Re-MAG3-HBP were
evaluated by ultrafiltration (Centrifree; Millipore, Billerica,
MA, USA). 186Re-MAG3-HBP (74 kBq in 33.3 μL saline)
was added to 300 μL of rat or human serum (or HSA, AGP
solution). Mixtures (280 μL) of 186Re-MAG3-HBP andserum were centrifuged at 1,000×g for 20 min at room
temperature. The radioactivity of the part of the initialsolutions (30 μL) and the filtrates (30 μL) was measured
with an auto-well gamma counter (ARC-2000; Aloka,
Tokyo, Japan). The free fraction and protein-binding ratio
were determined as follows:Free fractionð%Þ
¼ ðradioactivity of filtrateÞ=ðradioactivity of initial solutionÞ  100:
Proteinbinding ratioð%Þ ¼ 100  ðfree fractionÞ:The HSA (740 μM) and AGP (17 μM) solutions wereprepared at normal human serum concentrations.
In order to investigate the moiety with high affinity for
HSA in 186Re-MAG3-HBP, nonradioactive Re-MAG3 andbisphosphonate (1-hydroxyethylidene-1,1-diphosphonate;
HEDP) at concentrations of 0.4, 1, or 2.5 mM were added
to mixtures of 186Re-MAG3-HBP and HSA. Then, theEur J Nucl Med Mol Imaging (2009) 36:115–121

protein-binding ratios of 186Re-MAG3-HBP were evaluatedas described above.
The effects of each competitive protein-binding inhibitor
were examined in rat serum, human serum, and HSA
solution. Bucolome [20, 21], ceftriaxone [22], and cefazolin[23] as displacers of HSA binding site I and ibuprofen [13]as a displacer of HSA binding site II were added to each
serum at 400 μM before the addition of 186Re-MAG3-HBP.
Then, the protein binding of 186Re-MAG3-HBP was
evaluated as described above.
In vivo studies
Animal experiments were conducted in accordance with ourinstitutional guidelines; the experimental procedures wereapproved by the Kyoto University Animal Care Committee.
Biodistribution experiments were performed by intravenous
injection of 250 μL of the diluted tracer solution into male
Wistar rats (190–230 g, Japan SLC, Hamamatsu, Japan).
Ceftriaxone (50 mg/kg), as a competitive protein-binding
inhibitor, was administered intravenously just before thetracer was injected. Saline was administered to the rats in acontrol group instead of ceftriaxone. Groups of four or fiverats each were killed by decapitation at 5, 10, 30, 60, and
180 min postinjection. Tissues of interest were removed
and weighed. The entire left femur was isolated as arepresentative bone sample. The levels of radioactivity inthe tissues were determined with an auto-well gamma
counter and corrected for background radiation andphysical decay during counting.
To determine detailed blood time–activity curves, blood
samples of the male Wistar rats (190–230 g) were taken.
Specifically, a polyethylene cannula (inner diameter
0.5 mm, outer diameter 0.8 mm; Dural Plastics, Dural,
Australia) filled with heparin in saline was inserted into thefemoral artery of each rat anesthetized with sodium
pentobarbital. After intravenous injections via the tail ofthe tracer and ceftriaxone (50 mg/kg) or saline, blood
samples were taken at 2, 5, 7, 10, 15, 20, 30, 40, 50, and60 min postinjection. Blood samples were weighed, and theradioactivity was determined with an auto-well gamma
counter. The area under the blood time–activity curve wascalculated by numeral integration using a linear trapezoidal
formula from the time of tracer injection to the time of lastblood sampling and extrapolation to infinity based on amonoexponential equation [24].
Radiation dose estimates
For estimation of the radiation dose absorbed by the bone
and bone marrow, the level of activity in the bone marrow
was assumed to be 30% of that in the blood [25]. Red
marrow mass was assumed to be 25% of blood volume

117[25]. Blood volume was calculated using the following
formula [26].
Blood volumeðmLÞ ¼ 0:06  body weight ðgÞ þ 0:77
The bone mass of the rats was assumed to be 10% ofbody weight [27]. According to the International Commission on Radiological Protection, an equal distribution of theradionuclide to trabecular and cortical bones was assumed[28]. The no-decay-corrected activity from each source
organ was converted to a percentage of the injected dose
(ID). A time–activity curve was created for each organ byplotting the percentage of ID versus time. The area under anorgan’s activity curve from time zero to infinity wascalculated by the abovementioned method. According to
the values, absorbed radiation doses were calculated for an
adult patient using OLINDA 1.0 software (Vanderbilt
University) [29].
Statistical analysis
An unpaired Student’s t test was used for the biodistribution
experiments. A one-way analysis of variance followed byDunnett’s post-hoc test compared to the control group wasused for in vitro protein-binding experiments. Results wereconsidered statistically significant at p<0.05.

Results
Binding of 186Re-MAG3-HBP to serum protein
The percentages of 186Re-MAG3-HBP bound to rat serumand human serum were 97.4±1.1% and 97.0±0.3%, respectively. In the case of the purified human serum proteins,
186
Re-MAG3-HBP bound to HSA strongly (98.2±0.1%) andbound to AGP weakly (11.0±0.5%).
In the experiments in which nonradioactive Re-MAG3
or HEDP was added to a mixture of 186Re-MAG3-HBP
with HSA, Re-MAG3 appreciably inhibited the binding of
186
Re-MAG3-HBP to HSA on a dose-dependent basis(Fig. 1).
Effects of several drugs on the bindingof 186Re-MAG3-HBP to serum protein
The effects of several drugs as competitive inhibitors on
serum protein binding of 186Re-MAG3-HBP were evaluated. Data were expressed relative to the free fraction value
for the control group (without inhibitor). On the addition of
bucolome or ceftriaxone, the free fraction of 186Re-MAG3HBP in rat serum increased significantly compared withthat of the control (Fig. 2a). In human serum, the additionof ceftriaxone significantly increased the free fraction of

118

Eur J Nucl Med Mol Imaging (2009) 36:115–121**

2.5 mMa

Ibuprofen
Cefazolin

**

1 mM
*

0.4 mM

Ceftriaxone**

Bucolome*

Control0123
Relative Free Fraction
Re-MAG3

4

HEDP

Fig. 1 Displacement of 186Re-MAG3-HBP from protein in HSA. ReMAG3 and HEDP were loaded at 0.4, 1, and 2.5 mM (final
concentration). Data are expressed relative to the free fraction for thecontrol group (mean±SD for three experiments). Significant differences from control (without inhibitor) were identified with Dunnett’s
test (asterisk, p<0.05; double asterisk, p<0.01)

0

0.5
1
Relative Free Fraction

1.5bIbuprofen
Cefazolin

Ceftriaxone**

Bucolome
Control00.5
1
Relative Free Fraction

Ibuprofen*

186

Re-MAG3-HBP (Fig. 2b). In HSA solution, the additionof bucolome, ceftriaxone, cefazolin, or ibuprofen significantly increased the free fraction of 186Re-MAG3-HBP(Fig. 2c). The increase on the addition of bucolome,
ceftriaxone, or cefazolin was more remarkable than thaton the addition of ibuprofen (significance difference
determined with the Tukey–Kramer test).
In vivo studies
Table 1 lists the biodistribution of 186Re-MAG3-HBP withor without pretreatment with ceftriaxone in normal rats, expressed as a percentage of the ID per gram tissue (%ID/g).
As to the accumulation of radioactivity in the target tissue,bone, there was no difference between the ceftriaxonepretreated group and the control group. At the same time, thepretreated group had lower levels of radioactivity than did
the control group in nontarget tissues such as the kidney.
The blood time–activity curves of 186Re-MAG3-HBP
with or without pretreatment with ceftriaxone in rats,
expressed as %ID/g, are presented in Fig. 3. The clearance
value in the ceftriaxone-pretreated group and the control
group calculated from the area under the curve was 172.3±
11.8 and 115.8±14.4 mL/h, respectively.
Dosimetry
Table 2 lists the estimated absorbed radiation doses for 186ReMAG3-HBP with and without pretreatment with ceftriaxone.
We assumed that the radiation dose absorbed by theosteogenic cells is an index of the therapeutic effects. There
was no difference in the absorbed radiation dose versus thatwithout the ceftriaxone. Although the dose absorbed by red
marrow, a dose-limiting factor of bone-seeking radiopharmaceuticals, did not differ between the two groups, thatabsorbed by other nontarget tissues such as the kidney wasmarkedly decreased by pretreatment with ceftriaxone.

1.5

c
Cefazolin**

Ceftriaxone**

Bucolome**

Control00.5
1
Relative Free Fraction
Site II
Site I

1.5

Fig. 2 Displacement of 186Re-MAG3-HBP from protein in a rat
serum, b human serum, and c HSA. Each competitive protein-binding
inhibitor was loaded at a final concentration of 400 μM. Data areexpressed relative to the free fraction for the control group (mean±SDfor three experiments). Significant differences from control (without
inhibitor) were identified with Dunnett’s test (asterisk, p<0.05; double
asterisk, p<0.01)

Discussion
In the protein-binding experiments in vitro, 186Re-MAG3HBP showed very high ratios of protein binding in humanand rat serum. These high ratios of protein binding areprobably due to the strong binding of 186Re-MAG3-HBP toserum albumin because 186Re-MAG3-HBP exhibited extensive binding to HSA and little binding to AGP. In
experiments using several inhibitors of protein binding, the
HSA site I inhibitors were more effective than the HSA site
II inhibitor. A previous study showed that 99mTc-MAG3
binds specifically with binding site I on HSA [18]. If the
186
Re-MAG3 structure contributes to the binding of 186ReMAG3-HBP, the result of the experiments using an
inhibitor could be reasonable. Then, to investigate themoiety with high affinity for HSA in 186Re-MAG3-HBP,
the binding to HSA was examined when a large excess ofnonradioactive Re-MAG3 or bisphosphonate (HEDP) wasEur J Nucl Med Mol Imaging (2009) 36:115–121
Table 1 Biodistribution of radioactivity after intravenous
administration of 186ReMAG3-HBP with or without
pretreatment with ceftriaxone
in rats

Each value represents the mean
(SD) for four to five rats. Data
are expressed as percent of
injected dose per gram. Significance was determined by Student’s t test.
*p<0.05, **p<0.01

Tissue119
Time after administration
5 min

Pretreatment of ceftriaxone
Blood
0.98** (0.19)
Liver
0.32** (0.03)
Kidney2.76 (1.15)
Intestine
0.17* (0.03)
Spleen0.27 (0.03)
Femur
1.73 (0.20)
Muscle0.15 (0.02)
Control
Blood
1.32 (0.10)
Liver
0.44 (0.06)
Kidney4.35 (2.40)
Intestine
0.25 (0.07)
Spleen0.28 (0.02)
Femur
1.67 (0.22)
Muscle0.17 (0.02)

added to a mixture of 186Re-MAG3-HBP and HSA. As aresult, the addition of Re-MAG3 strongly inhibited thebinding of 186Re-MAG3-HBP to HSA, indicating that thebinding of 186Re-MAG3-HBP was attributable to the 186ReMAG3 moiety.
Ceftriaxone was the most effective of the HSA site I
inhibitors used in this study. At the same time, we consider
ceftriaxone, one of the antibiotics, to be effective forclinical applications because it can be administered clinically at a comparable dose from the point of view of safety.
Previously, Haradahira et al. reported that the uptake in thebrain of a radioligand for the glycine-binding site of the Nmethyl-D-aspartic acid receptor, [11C]L-703,717, was increased by the use of warfarin, which inhibits plasma protein
binding in mice [30]. The strategy is similar to that of ourstudy, but the ID of warfarin in that study was quite different
from a clinical dose. In this study, in vitro experiments were

% Injected Dose / Gram

10

10 min30 min60 min

180 min

0.57 (0.06)
0.13 (0.01)
1.22 (0.13)
0.09 (0.01)
0.12 (0.05)
1.88 (0.08)
0.12 (0.06)

0.19* (0.02)
0.08** (0.00)
0.85** (0.02)
0.08 (0.02)
0.06* (0.01)
3.47 (0.14)
0.06 (0.01)

0.05** (0.00)
0.07** (0.01)
0.62** (0.06)
0.14 (0.04)
0.05 (0.01)
3.89 (0.24)
0.01 (0.00)

0.02** (0.00)
0.06* (0.01)
0.58 (0.10)
0.26 (0.10)
0.05 (0.01)
4.01 (0.28)
0.01 (0.01)

0.77 (0.22)
0.16 (0.04)
1.56 (0.38)
0.10 (0.03)
0.15 (0.03)
1.91 (0.10)
0.11 (0.01)

0.27 (0.06)
0.09 (0.00)
1.40 (0.08)
0.10 (0.02)
0.08 (0.01)
3.43 (0.33)
0.06 (0.02)

0.07 (0.01)
0.07 (0.00)
0.85 (0.11)
0.11 (0.02)
0.05 (0.00)
4.15 (0.14)
0.01 (0.01)

0.03 (0.00)
0.08 (0.01)
0.73 (0.14)
0.30 (0.16)
0.04 (0.01)
4.14 (0.65)
0.02 (0.02)

performed under conditions where the final concentrationof inhibitors was 360 μM. It was reported that the earlyplasma concentration of ceftriaxone was over 400 μM after
a single intravenous administration of 2 g of ceftriaxone toan adult [31]. The administration of 2 g of ceftriaxone as aclinically available dose should be useful for the competitive
inhibition of protein binding because ceftriaxone significantly elevated the free fraction of 186Re-MAG3-HBP at thelower concentration in vitro. Another advantage of ceftriaxone is its long elimination half-life (6 to 9 h) [31, 32].
Maintaining a high concentration in the blood seems to befavorable for competitive inhibition at serum-binding sites.
Thus, in vivo experiments were performed with pretreatment of ceftriaxone as an inhibitor of protein binding. As
expected, the clearance from blood of 186Re-MAG3-HBP
was greater with the pretreatment than without. These
results suggest that ceftriaxone could be also useful in vivo
as a competitive protein-binding inhibitor. Furthermore, theceftriaxone-pretreated group also showed lower radioactivTable 2 Absorbed dose estimates of 186Re-MAG3-HBP with orwithout pretreatment with ceftriaxone

1

0.1

Organ

0

10

20
30
40
50
Time after Injection (min)60

Fig. 3 Radioactivity in blood after injection of 186Re-MAG3-HBP inrats. Ceftriaxone (open circles) (50 mg/kg) or saline (closed circles) as
a control was loaded just before 186Re-MAG3-HBP was injected. Data
are expressed as a percentage of the injected dose per gram (mean±SDfor seven rats) on a log scale

Osteogenic cellsa
Red marrowa
LiveraKidneysa
Small intestinea
Spleena
Effective dose equivalentb
Effective dosebab

Expressed as mGy/MBq
Expressed as mSv/MBq

Pretreatment with ceftriaxone

Control

8.75
3.01
0.03
0.42
0.01
0.02
0.69
0.50

8.87
3.05
0.04
0.77
0.01
0.03
0.72
0.51

120

ity levels than did the control group in nontarget tissues,
such as the kidney. These decreases in radioactivity might
be attributed to an enhanced distribution to the tissues withthe decrease in protein binding. However, there was no
significant change between the groups in the accumulationin bone. We suppose that it is the reason that 186Re-MAG3HBP binds to the hydroxyapatite in the bone and is retained
for a long term without excretion. Accordingly, pretreatment with ceftriaxone could be useful for improving thebiodistribution of 186Re-MAG3-HBP from the point ofview of decreasing the radiation dose to nontarget tissues.
The results from the estimates of absorbed doses indicatethat the dose absorbed by the kidney was certainly
decreased and that absorbed by osteogenic cells was almost
the same. We assumed that the dose of radiation absorbedby the osteogenic cells is an index of the therapeutic effects.
Although osteogenic cells should not be the exact target of
this type of internal radionuclide therapy, we cannot
describe a direct index of the therapeutic effect because
we used normal rats and calculated the radiation dose tonormal humans by extrapolation. The therapeutic (palliation) effects must occur by binding radiopharmaceuticals
with hydroxyapatite in or near the metastatic bone. Because
the accumulations of radiopharmaceuticals in the metastaticbone are not specific, it is predicted that the radiation doseof osteogenic cells would correlate with the therapeutic
effect. The results of the radiation dose indicate that it could
decrease toxicity to kidney tissue while obtaining the same
therapeutic effect. However, when the usual therapeutic
dose of 186Re-MAG3-HBP is injected, the strategy might
have little clinical merit because the initial radiation dose to
the kidneys is not very high. For example, when a muchhigher dose was injected with an autologous peripheral
blood stem cell transplant [33, 34], the strategy should be
more effective. At the same time, the dose absorbed by red
marrow, which is known as a dose-limiting factor of boneseeking radiopharmaceuticals, was not decreased. Sincealmost the entire dose absorbed by red marrow is from theradioactivity accumulated in bone, accelerated blood
clearance would not affect the absorbed dose in red marrow.
In conclusion, pretreatment with ceftriaxone enhanced
the clearance of the radioactivity of 186Re-MAG3-HBP inblood and nontarget tissues, although it had no effect on
accumulations in the bone. These findings suggested thattreatment with a protein-binding competitive inhibitor
would be effective for radiopharmaceuticals with highaffinity for serum protein to improve their pharmacokinetics, and this strategy might have even greater effects forother radiopharmaceuticals.
Acknowledgments This work was supported in part by a Grant-inAid for Scientific Research on Priority Areas from the Ministry of
Education, Culture, Sports, Science and Technology of Japan.

Eur J Nucl Med Mol Imaging (2009) 36:115–121

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